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首页> 外文期刊>African Journal of Biotechnology >Development of a novel real-time polymerase chain reaction (PCR) assay by amplification of double target genes for quantitative detection of Mycoplasma gallisepticum
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Development of a novel real-time polymerase chain reaction (PCR) assay by amplification of double target genes for quantitative detection of Mycoplasma gallisepticum

机译：通过扩增双靶基因定量检测鸡毒支原体，开发一种新型实时聚合酶链反应（PCR）检测方法

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Mycoplasma gallisepticum (MG) is the most pathogenic and economically significant pathogen in poultry worldwide. Detection of MG is the first and important step for controlling its transmission. A novel real-time polymerase chain reaction (PCR) assay targeting two conservative genes was developed and evaluated carefully in the study. The 16S rRNA and fMG-2 gene fragments were cloned into PCR 2.1 vectors, and recombinant plasmids (r16S21 and rFmg21) were evaluated by PCR, double digestion and nucleotide sequencing. SYBR green real-time PCR was developed using two purified plasmids as templates, and the amplification conditions and reaction systems of real-time PCR were optimized based on specificity, sensitivity and repeatability. A pair of standard curves were assembled for the real-time PCR by detecting two target genes 16S rRNA and fMG-2. The real-time PCR is highly specific to the target genes, with a detection limit of 9 copies/μl. The result of reproducibility shows that the real-time PCR remained consistent. The result of clinical samples demonstrated that the detection rate of the assay was significantly higher than that of the conventional PCR. The double target genes real-time PCR was highly specific, sensitive, and reproducible and could be used on clinical samples from commercial chickens.
机译：鸡支原体支原体（MG）是全世界家禽中最具致病性和经济意义的病原体。 MG的检测是控制其传播的第一步，也是重要的一步。开发了针对两个保守基因的新型实时聚合酶链反应（PCR）检测方法，并在研究中进行了仔细评估。将16S rRNA和fMG-2基因片段克隆到PCR 2.1载体中，并通过PCR，双消化和核苷酸测序评估重组质粒（r16S21和rFmg21）。以两个纯化的质粒为模板，开发了SYBR绿色实时PCR，根据特异性，敏感性和可重复性对实时PCR的扩增条件和反应体系进行了优化。通过检测两个靶基因16S rRNA和fMG-2，为实时PCR装配了一对标准曲线。实时荧光定量PCR对目标基因具有高度特异性，检测极限为9个拷贝/μl。重现性结果表明实时PCR保持一致。临床样品的结果表明，该测定的检测率显着高于常规PCR。双靶基因实时PCR具有高度特异性，灵敏性和可重复性，可用于商业鸡的临床样品。

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